1. Overall: The National Resource for Automated Molecular Microscopy Abstract The overall mission of the National Resource for Automated Molecular Microscopy (NRAMM) is to develop, test and apply technology aimed at automating and streamlining cryo-electron microscopy (cryoEM) for structural biology. Our goal from the outset was to provide a pipeline for molecular microscopy that puts it on a par with other structural techniques like X-ray crystallography, so that, once suitable samples are in hand, getting to a high resolution 3D map is a straightforward and rapid undertaking. This one time dream is now obtainable for well-behaved samples as a result of many innovations and improvements in hardware and software, including most recently the advent of a new generation of cameras that directly detect electrons and record high frame rate movies allowing for correction of sample movement during image exposure. There is still work to be done however in ensuring that this method can be applied to the most challenging and biologically interesting samples and serve a very large influx of new practitioners of this method. Our Technology Research and Development (TRD) Projects for the next five years are designed to meet the challenges. Our goals are to develop novel approaches and promote their widespread use, as well as to integrate complex technologies into an efficient and effective method. To achieve these goals, we will focus on three Technological Research and Development Projects that encompass both completely new approaches as well as dynamic evolution of our current technologies. In TRD#1, we will seek to address the critical need to improve and automate vitrified sample preparation; develop methods for time resolved vitrification; and greatly accelerate the throughput of negatively stained sample screening. In TRD#2, we will continue to optimize the performance of both high and mid-range instruments, particularly with regard to integrating and assessing the value of several major accessories; we will develop a high-throughput data acquisition pipeline to support negative stain screening; and we will continue to develop enabling tools for data assessment, processing, analysis and reconstruction. Finally, in TRD#3 we will embark on the development of a new automated and streamlined pipeline for support of in-situ molecular microscopy, that is visualizing molecular structures inside cells. The technological themes at the heart of this proposal support our mission of providing an automated and streamlined pipeline for molecular microscopy that provides data of the highest possible quality and promotes the accessibility of the method to the wider scientific community. This mission is driven by close interactions and feedback from Driving Biological projects (DBPs), and further tested and validated by Collaborative and Service Projects (CSPs). We will also continue to work to maintain excellence in the areas of training and dissemination to promote the broadest possible use of these technologies.